Abstract

BACKGROUND:

Plasmodium falciparum infection causes cerebral malaria (CM) in a subset of patients with anti-malarial treatment protecting only about 70% to 80% of patients. Why a subset of malaria patients develops CM complications, including neurological sequelae or death, is still not well understood. It is believed that host immune factors may modulate CM outcomes and there is substantial evidence that cellular immune factors, such as cytokines, play an important role in this process. In this study, the potential relationship between the antibody responses to the merozoite surface protein (MSP)-1 complex (which consists of four fragments namely: MSP-1(83), MSP-1(30), MSP-1(38) and MSP-1(42)), MSP-6(36) and MSP-7(22) and CM was investigated.

METHODS:

Peripheral blood antibody responses to recombinant antigens of the two major allelic forms of MSP-1 complex, MSP-6(36) and MSP-7(22) were compared between healthy subjects, mild malaria patients (MM) and CM patients residing in a malaria endemic region of central India. Total IgG and IgG subclass antibody responses were determined using ELISA method.

RESULTS:

The prevalence and levels of IgG and its subclasses in the plasma varied for each antigen. In general, the prevalence of total IgG, IgG1 and IgG3 was higher in the MM patients and lower in CM patients compared to healthy controls. Significantly lower levels of total IgG antibodies to the MSP-1(f38), IgG1 levels to MSP-1(d83), MSP-1(19) and MSP-6(36) and IgG3 levels to MSP-1(f42) and MSP-7(22) were observed in CM patients as compared to MM patients.

CONCLUSION:

These results suggest that there may be some dysregulation in the generation of antibody responses to some MSP antigens in CM patients and it is worth investigating further whether perturbations of antibody responses in CM patients contribute to pathogenesis.

Schematic representation of the MSP-1 and MSP-1/MSP-6/MSP-7 complex antigens used in the study. The schematic representation of the MSP-1 protein is shown (A). The MSP-1 precursor protein (i) undergoes proteolytic cleavage into four subunits as shown (ii). The MSP-142 molecule is further cleaved to MSP-133 and MSP-119 (iii). A proposed model (adapted from [23]) demonstrating the interactions of MSP-1 protein with the MSP-636 and MSP-722 molecules (B). The two allelic forms of MSP-183, MSP-130, MSP-138 and MSP-142 (D and F), in addition to MSP-636, MSP-722 and MSP-119, were expressed in E. coli and purified [49,50]. The purity of these recombinant proteins was examined by using 12% SDS-PAGE followed by Coomassie staining (C). Molecular weight (MW) is shown in kDa.

Prevalence of anti-MSP total IgG for the different subunits. The total IgG antibody response to the various MSP-1 subunits, MSP-636 and MSP-722 was measured by ELISA. The percentage prevalence was calculated as follows; (total number of positive sera/total number of sera tested) × 100. The prevalence of total IgG antibodies was statistically different between HC and MM for the indicated antigens (* = P < 0.05).

Levels of total IgG antibody responses to MSP-1 subunits, MSP-636 and MSP-722 antigens. Total IgG antibody responses were measured. Box plots depict median values with 25th- and 75th-percentile values represented by the bottom and top edges of boxes. Small diamonds indicate values that fall outside of the error bars. Only a few antigens demonstrated statistically significant differences (* = p < 0.05) when the antibody levels were compared among the different disease categories. The different subunits and allelic forms of MSP-1 are represented as d30, f30, d38, f38, d42, f42, d83 and f83.

Prevalence of anti-MSP IgG subclasses for the different subunits. The prevalences of the four IgG subclasses are shown. Test samples were considered positive if their OD values were higher than the mean plus two standard deviations of 10 pooled plasmas from malaria naïve donors from North America. The percentage prevalence was then calculated as described in figure 2.

Levels of IgG subclasses antibody responses to MSP-1 subunits, MSP-636 and MSP-722 antigens. The IgG1 (A) and IgG3 (B) antibody responses to the two allelic forms of each of the MSP-1 subunits, MSP-636 and MSP-722 were measured. The box plots depict median values with 25th- and 75th-percentile values represented by the bottom and top edges of boxes. Small diamonds indicate values that fall outside of the error bars. Statistically significant differences are depicted with the * (p < 0.05).

Correlation of antibody responses between the two dimorphic alleles, MSP-1D and MSP-1F. To determine if there was a correlation in antibody responses between the two allelic forms of MSP-1, a scatter plot of anti-MSP-1D versus MSP-1F total IgG antibody responses was generated. The lines represent the fitted values and the 95% confident interval lines. The Spearman correlation coefficients are shown in the upper left corner.